Development of biomolecular detection methods using kinase and nicking enzyme

Abstract

For the last decade, the in vitro diagnostic (IVD) market has steadily increased and is expected to grow at a fast-er rate in the near future. The most accurate technique among IVD techniques is molecular diagnosis to amplify and detect genes (DNA or RNA) directly related to disease and the most widely used IVD technology is personal glucose meter (PGM) that has been regarded as one of the most successful technology with high portability, low cost, simple operation, and reliable quantification. However, PCR-based molecular diagnostic technology is still limited for a wide-spread use because of the demand for thermal cycling system to amplify genes. In addition, utility of a PGM also is limited only for measuring glucose level. In this dissertation, an isothermal DNA amplifi-cation method that does not require a bulky and expensive thermal cycling system was devised by using the nicking enzyme. By employing this technology, uracil DNA glycosylase assay method was also developed. Fur-thermore, a cascade enzymatic reaction based on hexokinase and pyruvate kinase was developed to detect var-ious biomolecules utilizing PGM in a simple manner. The technologies described in this dissertation including the nicking enzyme-assisted isothermal DNA amplification method and kinase-based cascade enzymatic reaction system are expected to be able to cause social or economic big ripple effects in the field of future IVD.